Peptides, oligonucleotides and peptide nucleic acids are biologically important molecules and comprise polymers made up of distinct repeat units. In the case of peptides the repeat units are amino acids or their derivatives, while in the case of oligonucleotides the repeat units are nucleotides or their derivatives. Oligonucleotides can be further divided into RNA oligonucleotides and DNA oligonucleotides, as is well known to those skilled in the art, see for example P. S. Millar, Bioconjugate Chemistry, 1990, Volume 1, pages 187-191. In the case of peptide nucleic acids (PNA) the backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. The sequence of the amino acids in a peptide, the sequences of RNA nucleotides in RNA or DNA nucleotides in DNA, or the sequence of purine and pyrimidine bases in PNA, determine the function and effects of the compounds in biological systems.
The compounds are synthesised through coupling together their repeat units to give a specific sequence. The repeat units may be protected at one or more reactive sites using protecting groups, to direct coupling reactions to a specific site on the protected repeat unit. Deprotection reactions may be required after a coupling reaction to remove protecting groups and prepare the compound for a subsequent coupling reaction. Synthesis takes place in a sequence of cycles, each cycle comprising a coupling reaction followed by a deprotection reaction. Between reactions the removal of traces of excess reagents and reaction by-products to very low levels is necessary to prevent erroneous sequences being formed in the sequence of repeating units. When the coupling or deprotection reactions are carried out in liquid phase, this purification is often tedious, and is achieved by time consuming precipitation, crystallisation, or chromatography operations. The chemistries and methods available for coupling and deprotection of peptides, oligonucleotides and peptide nucleic acids have been well documented.
Peptide synthesis was revolutionised in 1963 by the advent of solid phase synthesis (Merrifield R B J Am Chem Soc 8.5, (1963) 2149). In this approach, the first amino acid in a sequence is bound to a resin bead. Subsequent amino acids are coupled to the resin bound peptide, and finally, when the desired peptide has been grown, it is cleaved from the resin. Importantly, at the end of each coupling or deprotection reaction, residual unreacted protected amino acids, excess reagents, and other side products can be removed by washing, including washing resin on a filter, or flushing a packed bed of resin with solvent. Solid phase peptide synthesis is now a standard technology for laboratory and commercial syntheses. The synthesis of oligonucleotides has followed a similar technological development to peptides, as described by Sanghvi, Y S, Org Proc Res & Dev 4 (2000) 168-169 and relies on solid phase synthesis in which a first oligonucleotide is linked to a solid phase. Further oligonucleotides are attached via cycles of coupling and deprotection reactions, with purification between the reactions carried out by washing, including washing a resin on a filter, or flushing a packed bed of resin with solvent.
Membrane processes are well known in the art of separation science, and can be applied to a range of separations of species of varying molecular weights in liquid and gas phases (see for example “Membrane Technology” in Kirk Othmer Encyclopedia of Chemical Technology, 4th Edition 1993, Vol 16, pages 135-193). Nanofiltration is a membrane process utilising membranes whose pores are in the range 0.5 to 5 nm, and which have MW cutoffs of 200-3,000 Daltons. Nanofiltration has been widely applied to filtration of aqueous fluids, but due to a lack of suitable solvent stable membranes has not been widely applied to separation of solutes in organic solvents. Ultrafiltration membranes typically have MW cutoffs in the range 1,000 to 500,000 Daltons. Recently new classes of membranes have been developed which are stable in even the most difficult solvents as reported in P. Vandezande, L. E. M. Gevers and I. F. J. Vankelecom Chem. Soc. Rev., (2008), Vol 37, pages 365-405. These may be polymeric membranes or ceramic membranes, or mixed matrix inorganic/organic membranes.
Diafiltration is a liquid filtration process in which a feed liquid containing at least two solutes is in contact with a membrane and is pressurised so that some fraction of the liquid passes through the membrane, wherein at least one solute has a higher rejection on the membrane than at least one other solute. Additional liquid is fed to the pressurised side of the membrane to make up for the liquid permeating through the membrane. The ratios between the concentration of the more highly retained solute and the concentration of the less retained solute in the permeate and retentate varies dynamically, increasing in the retentate and decreasing in the permeate.
The application of membrane separation to peptide synthesis has been reported for the re-concentration of peptides produced by biosynthesis as described by Tsuru T, Nakao S, Kimura S, Shutou T, Sep. Sci. and Technol., Volume 29, (1994), pages 971-984 or amino acid recovery from aqueous solutions as reported in Li S, Li C, Lui Y, et al., J. Mem. Sci., Volume 222 (2003), pages 191-201 or Wang X, Ying A, Wang W, J. Mem. Sci., Volume 196 (2002) pages 59-67. The use of membranes during peptide synthesis to separate growing peptides from excess reagents and reaction by-products was reported in U.S. Pat. No. 3,772,264. Peptides were synthesised in a liquid phase, with poly(ethylene glycol) (PEG) as a molecular anchoring group, and separation of the growing peptide chain from impurities was achieved with aqueous phase ultrafiltration. The separation required evaporation of the organic solvent after each coupling step, neutralisation followed by evaporation after each deprotection, and then for either coupling or deprotection, water uptake before ultrafiltration from an aqueous solution. Water was then removed by evaporation and/or azeotropic distillation before re-dissolving the PEG anchored peptide back into organic solvent for the next coupling or deprotection step. The complexity of the process makes it undesirable from a commercial perspective.